It is important in mammalian cell culture processes to maintain the physicochemical environment in view of dissolved oxygen, culture pH, temperature and shear sensitivity. Also the maintenance of the nutritional environment is important. The maintenance of the cultivation conditions limits the possibility to perform large scale culturing of mammalian cells. Especially concentration gradients can inhibit the cell growth of mammalian cells in large-scale bioreactors.
One of the objects of the present invention is to provide bioreactors and methods, which allow the cultivation of mammalian cells in large scale volumes. Furthermore, it is an object of the present invention to provide bioreactors and methods, which allow the cultivation of mammalian cells under optimal conditions, even if grown in large scale volumes and therefore allow a process performance and product quality independent of the size of the bioreactor.
It is an object of the present invention to provide large-scale bioreactors which allow the cultivation of mammalian cells in a homogenous environment with respect to process parameters such as pH, dissolved oxygen tension (DOT) and temperature, maintaining a well mixed cell suspension and blending nutrient feeds within the bioreactor.
Furthermore it is an object of the present invention to provide devices and methods which allow the production of mammalian cells and products of the mammalian cells, especially proteins, peptides, antibiotics or amino acids, synthesised by the mammalian cells, in a large-scale manner.
The present invention solves the technical problems underlying the present invention by the provision of bioreactors, bioreactor systems and methods for the cultivation of eukaryotic cells, especially of mammalian cells, according to the claims.
The present invention solves the technical problem underlying the present invention especially by the provision of a bioreactor for the cultivation of mammalian cells, characterised in that said bioreactor has at least two impellers. Furthermore, the present invention solves the technical problem underlying the present invention by the provision of a method for the cultivation and propagation of mammalian cells characterised in that at least one mammalian cell is cultivated under suitable conditions and in a suitable culture medium in a bioreactor, which has at least two impellers. Furthermore, the present invention solves the technical problems underlying the present invention by the provision of a bioreactor system for the cultivation of mammalian cells characterised in that a) a first bioreactor with a volume of at least 500 l is connected with b) a second bioreactor with a volume of at least 2000 l, which has a volume greater than the first bioreactor and wherein the second bioreactor with a volume of at least 2000 l is connected with c) a third bioreactor having at least two impellers and a volume of at least 10 000 l, which has a volume greater than the second bioreactor.
The present invention solves the technical problem underlying the present invention furthermore by the provision of a method to cultivate and propagate mammalian cells, characterised in that a) at least one mammalian cell is cultivated under suitable conditions and in a suitable culture medium in a first bioreactor with a volume of at least 500 l, b) the medium containing the cells obtained by propagation of the at least one mammalian cell is transferred into a second bioreactor with a volume of at least 2000 l, c) the transferred cells are cultivated in the second bioreactor with a volume of at least 2000 I, d) the medium containing the cells obtained in step c) is transferred into a third bioreactor with a volume of at least 10 000 l and e) the transferred cells are cultivated in the third bioreactor with a volume of at least 10 000 l.
According to the invention, the cultivated cells are eukaryotic cells, preferably animal cells, more preferably mammalian cells. The mammalian cells can be for example human cell lines, mouse myeloma (NS0)-cell lines, Chinese hamster ovary (CHO)-cell lines or hybridoma-cell lines. Preferably the mammalian cells are CHO-cell lines.
Preferably the cultivated cells are used to produce antibodies, more preferably monoclonal antibodies, and/or recombinant proteins, more preferably recombinant proteins for therapeutic use. Of course the cells may produce peptides, amino acids, fatty acids or other useful biochemical intermediates or metabolites. According to the invention the target concentration of the proteins produced by the cultivated cells is more than 0.5 g/l, preferably more than 2.0 g/l and most preferred more than 10.0 g/l. The method according to the invention can be used as a batch or in a fed-batch process. Although the cell-culture-medium used in the method according to the invention is preferably protein free medium, the design does not exclude the use of protein containing streams.